1. Field of the Invention
The present invention relates generally to the fields of bacteriology and mycology. More particularly, the present invention provides methods and compositions for increasing the effectiveness of existing antibiotics and antifungal agents and methods of overcoming bacterial and fungal resistance.
2. Description of Related Art
Gram positive organisms, particularly Staphylococci, Streplococci, and Enterococci, are increasingly seen as the major aetiological agents in infectious diseases. In the hospital setting, Staphylococcus aureus and Enterococcus faecalis account for more than 50% of isolates from blood stream infections (Cormican and Jones, 1996). In community-acquired infections, Streptococcus pneumoniae remains a leading cause of illness and death (Centers for Disease Control, 199). The ongoing and rapid emergence and spread of antibiotic resistance in these organism is thus a problem of crisis proportions.
One of the major impediments in treating Gram-positive infections is their limited susceptibility to fluoroquinolones, the latest addition to the arsenal of antibiotics. Since their introduction in the mid-1980s, fluoroquinolone antibiotics, have become the most used class of antibiotics in the world (Acar and Goldstein, 1997). One such antibiotic, ciprofloxacin (Davis et al., 1996), accounts for 90% of all quinolones used in medicine, Because of its spectrum of activity, oral availability, and relatively low cost, ciprofloxacin has been used for treating a wide range of infections, including those of unknown etiology. In 1996, three new indications for the use of ciprofloxacin were approved suggesting that the use of this antibiotic will continue for many years to come.
Although being highly active against most Gram negative microorganisms (MIC90 in the range of 0.1 xcexcg/ml), ciprofloxacin is less effective against Gram positive infections, particularly aerobic Gram positive cocci. The MIC90 values for S. aureus, E faecalis and S. pneumoniae are in the range of 1-5 xcexcg/ml, whereas the achievable tissue concentration of ciprofloxacin is only 4 xcexcg/ml (Davis et al., 1996). The high intrinsic resistance to ciprofloxacin, and the extensive use of quinolones both in human and veterinary medicine, has led to the emergence and dissemination of ciprofloxacin-resistant Gram-positive strains. This limitation has led to the quest for new, more effective fluoroquinolones.
Antibiotic resistance is mediated, at least in part, by the efflux of drugs from target cells by multidrug transporters (MDTs). These transporters promote the active efflux of a wide variety of drugs, including fluoroquinolone antibiotics, from the bacterial cells that are responsible for the particular infection. In 1991, Neyfakh et al. published the first description of a chromosomally-encoded bacterial multidrug transporter, Bmr, of the Gram positive bacteria Bacillus subtilis. Since then, practically every bacterial species analyzed, including pathogenic species such as Escherichia, Pseudomonas, Mycobacteria, etc. (Lomovskaya and Lewis, 1992; Poole et al., 1993; Takiff et al., 1996, reviewed in Nikaido, 1994; Lewis, 1994), has been shown to express one, or even several multidrug transporters. For example, B subtilis expresses at least three multidrug transporters, homologous Bmr and Blt (Ahmed et al., 1995) and an evolutionarily more distant Bmr3 (Ohki and Murata, 1997). Bmr and its close homolog in Staphylococcus aureus, NorA, promote the efflux of a variety of bacteriotoxic compounds, including ethidium bromide, rhodamine, acridines, tetraphenylphosphonium and puromycin, with fluoroquinolone antibiotics being one of the best transporter substrates (Yoshida et al., 1990; Neyfakh, 1992; Neyfakh et al., 1993). Importantly, drug efflux mediated by the Bmr and NorA transporters can be completely inhibited by the plant alkaloid reserpine, which by itself is not toxic to bacteria (Neyfakh et al.; Neyfakh, 1993).
Multidrug transporters also play an important role in both the intrinsic and acquired resistance of important fungal pathogens to antifungal agents. Particularly, multidrug transporters contribute to the resistance of Candida albicans, the fourth leading cause of all hospital-acquired infections, to azole antifungal agents.
There is little knowledge regarding the physiological role of multidrug transporters or the mechanism of their action; nevertheless these transporters appear to play an important role in the intrinsic resistance of bacterial cells to toxins and antibiotics. Inactivation of the chromosomal transporter genes usually leads to a dramatic increase in the sensitivity of bacteria to the transporter substrates (Poole et al., 1993; Ahmed et al., 1994; Okusu et al., 1996; Yamada et al., 1997). Disruption of the Bmr gene in B. sublilis, or the inhibition of the Bmr transporter with reserpine, reduces the minimal inhibitory concentration (MIC) of norfloxacin, a typical fluoroquinolone antibiotic, by a factor of five (Neyfakh, 1992). Similarly, multidrug transporters contribute significantly to the intrinsic fluoroquinolone resistance of Gram positive pathogenic cocci. Yamada et al. (1997) have recently shown that genetic disruption of the NorA gene increases the susceptibility of S. aureus to norfloxacin and ciprofloxacin by eight and four fold, respectively. Reserpine, which inhibits NorA-mediated drug efflux, reduces the MIC of norfiloxacin for wild-type S. aureus by at least four-fold (Markham and Neyfakh, 1996; Kaatz and Seo, 1995). Although the multidrug transporter of S. pneumoniae has not yet been identified, its existence is strongly supported by physiological data (Baranova and Neyfakh, 1997; Zeller et al., 1997; Brenwald et al.; 1997). Furthermore, reserpine has been shown to reduce the MIC of norfloxacin and ciprofloxacin for wild-type S. pneumoniae by the factor of 2-3 (Baranova and Neyfakh, 1997). In E. faecalis, the active efflux of fluoroquinolones has been demonstrated biochemically (Lynch et al., 1997) and, again, reserpine provides a two-fold increase in their susceptibility to fluoroquinolones.
In addition to being involved in the intrinsic resistance of Gram-positive cocci to fluoroquinolones, multidrug transporters contribute to the acquired resistance, which is selected upon exposure to these antibiotics. In S. aureus and S. pneumoniae, the acquired resistance has so far been attributed mainly to the sequential acquisition of mutations in the targets of fluoroquinolone action, topoisomerase IV and DNA gyrase (Cambau and Gutman, 1993; Ferrero et al., 1994; Munoz and De La Campa, 1996; Tankovi, 1996). From the limited studies of fluoroquinolone resistance mechanisms in E. faecalis, it appears that mutations of gyrase are present in at least some high level resistant isolates Korten et al., 1994). However, it has become apparent in recent years that these mechanisms of acquired resistance are complemented by over-expression of multidrug transporters. Such overexpression can result from either amplification of the transporter gene (Neyfakh, 1991); or mutations in the regulatory regions of these genes or regulatory proteins controlling their transcription (Ahmed et al., 1995; Kaatz and Seo, 1995).
Overexpression of the NorA multidrug transporter has been reported for strains of S. aureus selected for fluoroquinolone resistance both in vitro (Yoshida et al., 1990; Kaatz et al, 1990) and in vivo (Trucksis et al., 1991). From the discussion above it is clear that multidrug transporters present a major impediment to the treatment of Gram positive pathogenic insult. There exists a need for drug(s) that may circumvent these transporters to be useful in treatment regimens.
In order to meet the objectives of the present invention, there are provided methods of enhancing the antimicrobial action of antimicrobial agents by inhibiting the multidrug transporters in the microbes. A specific embodiment of the present invention contemplates a method for enhancing the antibacterial action of fluoroquinolones comprising contacting a bacterium with an inhibitor of NorA, wherein said inhibitor is an indole, a urea, an aromatic amide or a quinoline.
In more particular embodiments, the inhibitor is an indole that has the general formula: 
wherein R1 is phenyl, 2-naphthyl, o-anisole, R2 is H or CH3, R1 and R2 are two naphthyl groups fused to the indole ring, R3 is H, R4 is NO2, SO3H, NH2 and CF3 or CCl3, R5 is H, and R6 is H. More particularly, the R1 may be a phenyl group and R4 may be SO3H or NO2. In other specifically preferred embodiments, the R1 may be 2-naphthyl and R4 may be CCl3 or CF3. In still additional embodiments, the R1 may be o-anisole and R4 may be NO2. In further embodiments, the R1 and R2are two naphthyl groups fused to the indole ring. Additional preferred embodiments are contemplated in which R1 is phenyl and R2 is CH3.
In those aspects of the invention in which the inhibitor is a urea, the urea may have the general formula: 
wherein R1 is OR, Br, Cl, or F, R2 is OR, NHCO2R, Cl, F, or H, R3 is Cl, Br, OR, or CO2R, R4 is Cl or Br, R5 is H, R6 is H, R7 is H, R8 is a conjugated or aromatic system, R9 is H, OR, Cl or Br, R10 is H, OR, or Cl. More particularly, R1 may be OMe, and either R3 or R4 may be Cl, in addition to R8 being C(xe2x95x90O)Ph or a fused aromatic ring at R7-R8.
In those embodiments in which the inhibitor is an aromatic amide, the inhibitor may have the general formula: 
wherein R1, R4 and R5 are H, R2 and/or R3 are small electron-withdrawing groups, and R6 is a substituted or unsubstituted alkyl of at least six atoms including O, N or S, with or without a phenyl ring. More particularly, the electron-withdrawing group is selected from the group consisting of Cl, and F. In other preferred embodiments, the R4 and R6 in the aromatic amide of structure III are smaller conjugated systems of 2-6 atoms of C, O, N or S, and includes a phenyl ring.
In those embodiments in which the inhibitor is a quinoline, the inhibitor may have the general formula: 
wherein R2 may be 3,4-dimethoxybenzene or p-toluene, R3 is H, R4 may be CO2R, C(xe2x95x90O)NH2, or H, R5 is H, R6 is H, NO2, SO3H, NH2, CF3 or CCl3, R7 group, NO2, SO3H, NH2, CF3 or CCl3 and R8 is H. In particular, the combination where R2 is 3,4-dimethoxybenzene, R3 is H, R4 is CO2R, R5 is H, R6 is H, R7 is Me, and R8 is H.
It is particularly contemplated that the bacterium is Streptococcus pneumonia, Enterococcus faecalis, Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Enterococcus faecalis, Staphyloccus aureus, Streptococcus pyogenes, Escherichia coli, Serratia marcesens. Of course, those of skill in the art will realize that the inhibitors found to be useful in applications against these bacteria also may be useful against other bacterial infections. As such these are exemplary bacteria and the present invention is not intended to be limited to infection caused by these bacteria.
Another aspect of the present invention provides an indole having the general formula: 
wherein R1 is phenyl, 2-naphthyl, o-anisole, R2 is H or CH13, R1 and R2 are two naphthyl groups fused to the indole ring, R3 is H, R4 is NO2, SO3H, NH2 and CF3 or CCl3, R5 is H, and R6 is H. In specific embodiments, R1 is phenyl and R4 is SO3H or NO2. In other embodiments, R1 is 2-naphthyl and R4 is CCl3 or CF3. In still additional embodiments, R1 is o-anisole and R4 is NO2. Other embodiments contemplate an indole in which R1 and R2 are two naphthyl groups fused to the indole ring. Yet another indole molecule contemplated is one in which R1 is phenyl and R2 is CH3.
Also contemplated herein is a urea having the general formula: 
wherein R1 is OR, Br, Cl, or F, R2 is OR, NHCO2R, Cl, F, or H, R3 is Cl, Br, OR, or CO2R, R4 is Cl or Br, R5 is H, R6 is H, R7 is H, R8 is a conjugated or aromatic sy R9 is H, OR, Cl or Br, R10 is H, OR, or Cl. More particularly, R1 may be OMe, and either R3 or R4 may be Cl, in addition to R8 being C(xe2x95x90O)Ph or a fused aromatic ring at R7-R8.
Also contemplated herein is an aromatic amide having the general formula: 
wherein R1, R4 and R5 are H; R2 and/or R3 are small electron withdrawing groups, and R6 is substituted or unsubstituted alkyl of at least six atoms including C, O, N or S, with or without a phenyl ring. Specifically the aromatic amide may be one in which R4 and R6 are smaller conjugated systems of 2-6 atoms of C, O, N or S, and includes a phenyl ring.
Another aspect of the present invention provides a quinoline having the general formula: 
wherein R2 may be 3,4-dimethoxybenzene or p-toluene, R3 is H, R4 may be CO2R, C(xe2x95x90O)NH2, or H, R5 is H, R6 is H, NO2, SO3H, NH2, CF3 or CCl3, R7 is an alkyl group, NO2, SO3H, NH2, CF3 or CCl3 and R8 is H. In particular, the combination where R2 is 3,4-dimethoxybenzene, R3 is H, R4 is CO2R, R5 is H, R7 is Me, and R
Another aspect of the present invention contemplates a method of screening for inhibitors of NorA comprising providing a cell expressing only a single functional transporter, said transporter being Nor A; contacting said cell with a transportable element in the presence of a candidate inhibitor substance; and comparing the transport of said element by said cell with the transport of said element in the absence of said candidate inhibitor substance.
In particularly preferred embodiments, the cell is a bacterial cell. In additional preferred embodiments, the bacterial cell is a Gram negative bacterial cell. In other preferred embodiments, the bacterial cell is a Gram positive bacterial cell. More particularly, the Gram positive bacterial cell is a Bacillus subtilis cell. In specific embodiments, it is contemplated that the B. subtilis cell contains disrupted Bmr and Bit genes.
In other preferred embodiments, it is contemplated that the NorA is Staphyloccus aureus NorA, Streptococcus pneumoniae multidrug transporter, or Enterococcus faecalis multidrug transporter. In particular embodiments the transportable element is ethidium bromide. In other embodiments, the transportable element is a fluoroquinolone.
Another aspect of the present invention provides a method for treating a subject with a bacterial infection comprising providing to said subject a fluoroquinolone and an inhibitor of NorA, wherein said inhibitor is an indole, a urea or an aromatic amide. In preferred embodiments, the bacterium is Streptococcus pneumonia, Enterococcus faecalis, Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermis, Mycobacterium smegmatis and Serratia marcesens. 
Also provided herein is a pharmaceutical composition comprising a fluoroquinolone and an inhibitor of NorA, wherein said inhibitor is an indole, a urea or an aromatic amide. In certain embodiments, the fluoroquinolone is selected from the group consisting of Sparfloxacin, Levofloxacin, Grepafloxacin, Temafloxacin, Clinafloxacin, Bay 12-8039, Trovafloxacin, DU6859a, Sarafloxacin. In addition to the fluoroquinolones, it is contemplated that other quinolones such as fluoronaphthyridones may be useful in the compositions of the present invention. A particularly preferred quinolone is LB20304. Of course, one of skill in the art will realize that there will be other antibacterial fluoroquinolones that may be combined with the inhibitors of the present invention. As such, the present invention is not limited for use in compositions with the listed fluoroquinolones alone, rather the inhibitors will be useful in combination with any fluoroquinolone or other agent that possesses antibacterial activity. Additionally, the inhibitors of the present invention will be useful with any antibacterial agent which is or would be effective at killing, reducing or otherwise diminishing the growth of bacteria but for the presence of resistance created by the multidrug transporters in such bacteria.
Another aspect of the present invention describes a method of enhancing the antifungal action of azole antifungal agents comprising contacting a fungus with an inhibitor of a fungal multidrug transport protein, wherein said inhibitor is an indole, a urea, an aromatic amide or a quinoline. More particularly, the indole has the general formula I, the urea has the general formula II, the aromatic amide has the general formula III and the quinoline has the general formula XV. It is particularly contemplated that the fungus is from a species selected from the group consisting of Candida, Cryptococcus, Blastomyces, Histoplasma, Torulopis, Coccidioides, Paracoccidioides and Aspergillis. Of course one of skill in the art will realize that the invention is not limited to only treating these fungal infections but rather that the inhibitors will likely be useful against many other fungal species.
Yet another embodiment of the present invention provides a method of screening for inhibitors of a fungal multidrug transporter comprising: providing a cell expressing only a single functional transporter, said transporter being fungal multidrug transporter; contacting said cell with a transportable element in the presence of a candidate inhibitor substance; and comparing the transport of said element by said cell with the transport of said element in the absence of said candidate inhibitor substance. In specific embodiments, the cell is a fungal cell.
In specifically preferred embodiments, the cell is from the Candida species. In other preferred embodiments, the multidrug transporter is a Candida multidrug transporter. In certain embodiments, the antifungal agent is a triazole antifungal agent. In other preferred embodiments, the triazole is selected from the group consisting of ketoconazole, miconazole, itraconazole, fluconazole, griseofluconazole, clotrimazole, econazole, terconazole and butaconazole. It should be understood that these triazole anti-fungal agents are exemplary agents, additional azoles also may be useful in the present invention. Such additional azoles may be derived form these azoles listed or have a similar mode of action to these compounds.
Another aspect of the present invention provides a method of treating a subject with a fungal infection comprising providing to said subject an azole antifungal agent and an inhibitor of a fungal multidrug transport protein, wherein said inhibitor is an indole, a urea, an aromatic amide or a quinoline. In specific embodiments, the antifungal agent is selected from the group consisting of ketoconazole, miconazole, itraconazole, fluconazole, griseofluconazole, clotrimazole, econazole, terconazole and butaconazole.
Also provided herein is a pharmaceutical composition comprising an azole antifungal agent and an inhibitor of a fungal multidrug transporter, wherein said inhibitor is an indole, a urea, or an aromatic amide.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.